The mechanism by which metal ion requiring nucleases catalyze the hydrolysis of DNA is not well characterized, because the structural complexity of the substrate prohibits a detailed chemical study of the reaction. To provide insight into these complex reactions, we are studying the chemical mechanism by which bacterial enzymes hydrolyze structurally simple phosphodiesters and phosphorothioates. A phosphohydrolase from Enterobacter aerogenes, which catalyzes the hydrolysis of alkyl mono- and diesters of phosphoric acid and esters of thiophosphoric acid, requires divalent metal ions for activity, and we propose to determine the role of the metal in catalysis. We are studying this enzyme by direct measurement of metal binding, spectroscopic studies to determine the relationship between the metal and its binding site and the metal and the substrates, and stereochemical studies to establish whether a covalent intermediate between the enzyme and substrate is involved. We also propose to study a phosphotransferase from E. aerogenes which apparently catalyzes both phosphotransferase and phosphohydrolase activities without the participation of a covalent intermediate; we plan to determine whether a metal ion is required for activity and establish whether covalent catalysis is involved. In addition, we plan to compare the structures of these enzymes so that we might determine whether they are structural and genetic variants and identify why their catalytic properties differ.